Nerve agents and a large number of pesticides fall into a class of chemicals called organophosphates (OP), which pose serious health concerns for military and civilian populations alike. OP-pesticide compounds are commercially available and abundantly used worldwide. In addition to hazards associated with routine, chronic, low-level exposure over time, misuse of OP-pesticides has also led to malicious, suicidal, or accidental acute intoxications; the repercussions of which (chronic and acute alike) lead to life-long and debilitating neurological deficits. Although OP pesticide use in the United States is declining, malathion is widely used in agriculture, residential landscaping, public recreation areas, and in public health pest control programs such as mosquito eradication (US EPA, 2007, Bonner 2007). Forty OP pesticides are registered in the U.S., with at least 73 million pounds used in agricultural and residential settings (Maugh 2010). According to the World Health Organization (WHO), more than one million serious accidental, and two million suicidal poisonings with pesticides occur worldwide every year (Jeyaratnam 1990). In rural regions of developing countries, suicide via OP pesticides kills ˜200 000 people per year. Unintentional poisoning kills fewer people, but is a still problem where highly toxic OP pesticides are available (Eddleston 2008).
In addition to OP-pesticides, tactical use of OP-nerve agents (chemical weapons, such as sarin, soman, Tabun, and VX to name a few) against the warfighter as well as non-military personnel is a potentially imminent and legitimate deadly threat. Radical political groups and unstable governments have successfully deployed nerve agents in the recent past. Tabun was used by the Iraqi military against Iran in the 1980s, and sarin was used by the terrorist group Aum Shinrikyo in the Matsumoto and Tokyo subways in the mid-1990s (Nozaki H 1995) (Macilwain 1993) (Brown M A 1998). The United Nations investigation of allegations against the Syrian Arab Republic confirmed “unequivocally and objectively” that chemical weapons were used in the Ghouta area of Damascus in Syria on Aug. 21, 2013 (Sellstrom A 2013). During these events, surface-to-surface rockets containing the nerve agent sarin were utilized. Within three hours of the attack, three hospitals in the Damascus area received approximately 3,600 patients displaying symptoms consistent with nerve agent exposure. From this event, it is estimated that there were approximately 1429 deaths, 426 of which were children (The White House 2013). Most recently, the International Business Times reported (Feb. 22, 2015) that, “Islamic State Fighters in Libya may have seized large amounts of chemical weapons, including mustard gas and the nerve agent Sarin.” “The ISIS Fighters near Tripoli have begun testing the weapons that reportedly once belonged to the former regime of Moammar Gadhafi. ‘Before his death, Gadhafi left approximately 1,000 cubic tons worth of material used for manufacturing chemical weapons and about 20,000 cubic tons of mustard gas.’” Taken together, in addition to the pervasive use of dangerous OP pesticides worldwide, it is also highly plausible that OPs may be used again in the future with malicious intent.
The prolific use of OP-pesticides worldwide coupled with a serious homeland security concern that OP-based nerve agents may be deployed either against our troops or civilians on our home soil has stimulated the search for rapid, miniaturized and inexpensive point-of-care diagnostic devices for first responders and medical personnel to detect the degree of exposure (Langenberg 2009). Taken together, there is an unmet need to rapidly identify patients that have been exposed to OP toxicants and determine degree of exposure, particularly at lower exposure levels. Lower OP exposure produces vague, nondescript signs and symptoms that are not easily differentiated from other conditions. The lack of an accurate diagnosis of some exposure to OP agents may result in a delay, or complete lack of receipt of needed treatment, the use of life-endangering resuscitation drugs, or the use of high risk anesthetics that can cause injury or death in those who have been exposed to OP agents. In the event of a military or terrorist release of a chemical warfare nerve agent, the analysis of biomedical samples for the presence of biomarkers to confirm exposure is imperative to ensure that appropriate medical countermeasures are administered in a timely manner. In addition to identifying those with true nerve agent poisoning (both apparent and subclinical, also referred to as the “walking wounded”), it is important to verify non-exposure to reassure worried civilian or military personnel (also referred to as the “worried well”) (Sambursky 2015).
Specific diagnostics to identify exposure to OPs were rapidly developed following the attacks in Japan in the mid-1990s. Assays that measured metabolites to identify the exact OP agent relied on acid hydrolysis products in urine. However, because OP compounds are unstable in pure aqueous solutions and are rapidly degraded upon entering the body, the urinary metabolites are excreted very rapidly and are not practical for retrospective OP exposure analysis (Driskell 2002) (Shih 1994). Therefore, analysis for the presence of intact OP agents in blood is inappropriate unless samples are collected immediately after exposure.
However, upon entering the body via inhalation or skin absorption, OP agents enter the blood and immediately combine with blood proteins to form blood protein-nerve agent adducts. Specifically, OPs irreversibly complex with both acetylcholinesterase (AChE) and butylcholinesterase (BChE) enzymes. AChE and BChE enzyme activity are good biomarkers for risk assessment because they not only substantiate exposure, but also directly provide a quantitative biochemical effect of the exposure (i.e., enzyme inhibition). Furthermore, these biomarkers of exposure are persistent at least 16 days post-exposure, likely much longer (Fidder 2002) (Solano 2008).
Numerous methods have been developed to assess AChE and/or BChE levels, including the Ellman assay, fluorescence assay, electrochemical assay, Michel (ΔpH) ChE assay, radioactive assay, and Walter Reed Army Institute of Research (WRAIR) assay. The Model 400 Test-Mate™ ChE assay kit, which uses enzyme activity for screening of OP exposure is FDA-approved and commercially available. All of these methods are based on detecting a meaningful decrease in enzyme activity from a previously established baseline, which can vary depending on the testing method (Haigh J R 2008).
AChE is a protease found primarily in the brain and red blood cells and is responsible for breaking down the neurotransmitter acetylcholine (ACh) at the cholinergic synapses in the Central and Peripheral Nervous Systems (CNS/PNS) and at neuromuscular junctions. Inhibition of AChE by an OP leads to the accumulation of ACh and excessive stimulation of the CNS/PNS. When ACh levels reach a critical threshold, classic symptoms of cholinergic crises appear such as miosis, nausea, vomiting and diarrhea, hypersecretions, difficulty breathing, and death in extreme cases.
The diagnostic described herein seeks to capitalize on the finding that the degree of AChE or BChE inhibition is directly proportional to the extent of OP-exposure. The AChE enzyme has three isoforms produced by alternative splicing of pre-mRNA: synaptic isoform (AChE-S) is produced and found primarily in brain and muscle tissues, erythrocytic isoform (AChE-E) is anchored to red blood cell (RBS) membranes. Since it is very invasive to obtain AChE-S, measurement of AChE-E is usually sampled as a surrogate indicator of AChE inhibition in the CNS/PNS (Knechtges 2008). BChE is readily found in plasma but the biological role of BChE is not fully understood. Although BChE also hydrolyzes ACh, it is primarily recognized as a scavenging enzyme for detoxification of naturally-occurring compounds (Lotti 1995) (Poet T S 2003) (Shen Z 2004).
In the past several years, numerous groups have independently demonstrated that there is a statistically significant correlation of BChE inhibition to measurable signs and symptoms of OP intoxication as scored by various poisoning classification systems (e.g. Glasgow Coma Scale, APACHE II, Proudfoot classification, and Peradenya Organophosphorus Poisoning Scale) (Prasad D R M M 2013, Kumar C U 2014, Ahmed K M 2014, Patil G 2015, Patil S L 2014, Sungertekin, H 2006) Additionally, BChE is easily extracted and measured from plasma (Li 2010), has a high rate of reaction, and is vastly more abundant (10-500 times) than AChE (Nigg H N 2000) (Holland K D 2008) (Solano 2008). Taken together, either AChE or BChE serves as an excellent direct or indirect biomarker, respectively, for OP exposure (Prasad DRMM 2013), (Haigh J R 2008).
However, measurement of cholinesterase (both AChE and BChE) is complicated by inter- and intra-individual variability of ChE activity and relationship to clinical signs and symptoms. The variability of AChE activity between individuals based simply on genetics, sex, race, or age is estimated as high as 23% (Lessenger J E 1999). The deviation of BChE from mean is up to 65% (Knaak J S 2012). This level of cholinesterase (ChE) population variation underscores the importance of having individual baseline values of ChE activity to interpret results when assessing exposures to ChE-inhibitors. In the absence of a baseline (which is likely the case in the event of a mass casualty scenario) ChE inhibition measurements are often based on a population average that introduces a large degree of uncertainty caused by the enzyme variability within the population. These uncertainties reduce the sensitivity of AChE/BChE activity as a biomarker and thus may provide ambiguous results for subclinical, low-dose OP exposure (<20% enzyme inhibition). However, cholinesterase inhibition may be subclinical in appearance only, as there is evidence that even a low dose exposure of OP neurotoxicants can cause long-term, significant chronic neurological deficits (McDonough J H 1997) (Abou-Donia 2003) (Chen 2012).
Recent advances in the application of nanomaterials as transducers, recognition agents or labels, and advances in immunoassay techniques are now being exploited in the design of improved devices for the biomedical detection of non-metabolized OP pesticides and nerve agents, metabolites, and protein adducts (Black R M 2013). Several laboratories have developed techniques that approach meeting various diagnostic needs of OP exposure, but each method is caveated with drawbacks. Some recent examples include:    Zhang et al., (2013) developed a lateral flow assay test strip that detects OP-AChE adducts by selectively capturing the AChE with quantum dot (Qdot)-tagged anti-AChE antibodies and zirconia nanoparticles (ZrO2). A sandwich of Qdot-Anti-AChE Ab/OP-AChE/ZrO2 was detected using a hand-held fluorescence detector. Lu et al. (2011) also utilized ZrO2 nanoparticles to bind BChE from plasma in a disposable electrochemical immunosensor. The drawback to both of these approaches is that only OP-bound AChE or BChE levels were detected yielding a quantitative response that, in the absence of a baseline, are challenging to interpret (Zhang W 2013).    Du et al. and Ge et al. (2011 and 2013) developed both an integrated lateral flow test strip with an electro-chemical sensor (LFTSSES) and an electrochemical method using Fe3O4/Au nanocomposites resulting in rapid, selective, and sensitive quantitation of OP exposure based on parallel measurements of both OP-AChE and reactivated AChE (using treatment with the oxime 2-PAM). Even though these methods attempted a baseline-free approach, both systems rely on the reactivator (oxime) to strip the OP from the AChE molecule to generate baseline levels of enzyme. These approaches are not compatible for all OP exposures in which the phosphorylated AChE or BChE can either spontaneously regenerate, or have aged to the point of permanent OP-adduction (Du D 2011) (Ge X. 2013).    Dr. Rudolph Johnson laboratory developed and optimized a baseline-free approach to measuring OP-bound and free BChE using immunomagnetic separation couple with liquid chromatography tandem mass spectrometry (LC-MS/MS). The optimized method captures >88% of the BChE in a specimen. The drawback to this method is that it is laboratory-based requiring trained personnel to perform specialized sample extractions and operation of sophisticated instruments (Knaak J S 2012) (Pantazides B G 2014).    Test-mate ChE Cholinesterase Test System (Model 400) is a commercially available product from EQM Research, Inc. for determining the percentage of active Acetylcholinesterase or Butyrylcholinesterase. The system utilizes a microwell format with a small fixed wavelength photometer to read results. The test method is based on the conventional Ellman assay which has the innate drawback of the requirement of a baseline result in order to accurately determine the degree of cholinesterase inhibition in a test subject. A secondary drawback of the system for point of care field use is that it is designed for use in a clinical laboratory by a medical technician under supervision of a laboratory director.